The objective of the proposed research is to develop a molecular genetic system for study of metabolic regulation at the level of compartmentation in eucaryotic cells. Specific goals are the isolation and characterization of genes encoding isozymes which catalyze shuttle cycle reactions on opposite sides of the mitochondrial membrane. This proposal focuses on the genomic sequences from Saccharomyces cerevisiae which encode mitochondrial and cytoplasmic malate dehydrogenases. These enzymes, with respective functions in the tricarboxylic acid cycle and in gluconeogenesis, regulate the partitioning of critical reducing equivalents and substrates between compartments. The isozymes of malate dehydrogenase will be purified from yeast for determination of partial amino acid sequences. Oligodeoxynucleotide probes will be chemically synthesized on the basis of the primary protein structures and used to identify homologous genomic sequences in recombinant plasmid libraries of yeast DNA. The isolated sequences will be characterized by partial nucleotide sequence analysis to confirm the identity of genes encoding mitochondrial and cytoplasmic malate dehydrogenases and by restriction endonuclease and transcription mapping. To begin to define essential metabolic functions, the coding region of each isolated gene will be interrupted by insertion of a second selectable DNA sequence in vitro. These altered sequences will be used to replace corresponding genomic sequences in vivo by yeast transformation and the phenotypes of resulting yeast mutants defective in expression of mitochondrial or cytoplasmic malate dehydrogenase analyzed in detail. These studies will form the basis of a molecular genetic system for future efforts aimed at defining (a) eucaryotic genetic determinants or enzyme function, cellular localization, and expression and (b) in vivo functions of compartmentalized isozymes in coordinating central metabolic pathways separated by the mitochondrial membrane.